Evaluation of an enzyme-linked immunosorbent assay for the detection of Mycoplasma hyopneumoniae antibody in porcine serum RF SHELDRAKE* AND LF ROMALIS Elizabeth Macarthur Agriculturh Institute, PMB 8, Camden, New South Wales 2570 SUMMARY: An enzyme-linked immunosorbent assay (ELISA) for detecting antibody to Mycoplasma hyopneumonlae in porcine serum is described. The results are presented as an ELiSA ratio, calculated by dividing the absorbance of the test sample by the mean absorbance of control negative sera. In known infected pigs, the ELISA ratio was highest when the serum concentration applied t o the ELISA plate was diluted 1 in 20 In PBS Tween. Mean ELlSA ratios ranged from 1.2 f0.3 for pigs without porclne enzootlc pneumonia (PEP) lesions to 5.5 f 1.5 for pigs observed with a PEP lesion reacting positively with immunofiuorescent histopathoiogy. Pigs observed with typical PEP lesions at slaughter, but not confirmed by immunofiuorescent histopathology had a mean ELiSA ratio of 4.9 f 1.7. The ELlSA was highly sensitive (95.6%) and specific (98.8%) when pig sera from commercial piggeries of known Mhyopneumoniae infection status were assessed. No cross-reactivity with serum from a pig hyperimmunised with killed M flocculare was detected, and reactivity with serum from another pig hyperimmunised with killed Mhyorhlnfs showed only weak cross-reactivity, which failed to reach the ELISA positive threshold (ELISA ratio 3)for Mhyopneumonlae. Aust Vet J 69: 255 - 258
-
Introduction Enzootic pneumonia caused by Mycoplasma hyopneumoniae is a disease of major economic importance to pig production worldwide (Backstrom andHoefling 1982;Muirhead 1987). One of the difficulties in effectively controlling the disease has been the lack of a simple and cost effective means of monitoring progress of the disease in the herd. Complement fixation tests (Boulanger and L'Ecuyer 1968; Roberts and Little 1970; Etheridge and Lloyd 1980) and indirect haemagglutination tests (Goodwin et a1 1969; Lam and Switzer 1971) have been used although problems with specificity and sensitivity have been reported (McKean et al 1979). More recently enzyme-linked immunosorbent assays (ELISA) have been used (Bruggmann et al 1977; Nicolet et a1 1980; Freemanet a1 19W, Kobisch andNicolet 1987;Eberli 1987; Mori et a1 1987; ). The antigens that were used in these assays were prepared according to the methods of Bruggmann et a1 (1977) or Nicolet et uZ(1980) by the addition of a solubilising detergent to a suspension of whole cells that was semi-purified by column chromatography. Kazama et a1 (1989) used a Tween 20 solubilised fraction, which was prepared by molecular sievingt. These workers found superior specificity compared with the method of Nicolet el a1 (1980). Feld et a1 (1990) evaluated a monoclonal blocking ELISA and found high specificity when compared with an indirect haemagglutination assay. Our aim was to evaluate an ELISA that used solubilised and purified antigens, and to determine the optimum conditions under which the ELISA might be used as a routine diagnostic test.
Materials and Methods Antigens The antigens used in the ELISA were prepared according to the procedures of Auspharm International* and are commercially available through Auspharm. They were obtained from a culture ofM hyopneumoniae (J Strain) which was cultured in broth (Friis 1977) by rotating continuously at 37°C until the change in pH resulted in the medium being honey coloured (pH 6.9 - 7.2). The broth culture was centrifuged at 10 000 g for 20 min. The pellet was subsequently washed 3 times in Tris buffered saline (TBS) (pH 7.4) in a similar manner. After the fmal wash the pellet was resuspended in 10 ml TBS, and sonicated for 30 s in a sonicatorO using a 50% duty cycle, output 3.5. The sonicate was then centrifuged and the pellet Collected and resuspended in TBS. After equilibration in TBS containing 0.2% sodium dodec 1 sulphate (SDS) the material was further purified on a column .
!?
The peak collected between 360 and 405 ml was pooled and concentrated. The antigen was titrated from 1/50 to 1/2OOO and at a titration of 1/1000 the ELISA reaction maximised the response for sera dilutions ranging from 1/20 to 1/250. The antigen was applied to the ELISA plate in a volume of 100 11. The procedures for coating and reading the plates have been described previously (Sheldrake el al 1990).
Animals To provide hyperimmune serum, and serum before and after infection with M hyopneumoniae, six Landrace x Large White pigs6 to 8weeksofagewereobtainedfromacomercialpigge1y
* Present address: Division of Animal Industries, NSW Agriculture, Locked Bag 21, Orange, New South Wales 2800 Sephacryl@,Pharmacia Chemicals, Copenhagen, Denmark
Australian Veterinary JOWMI Vol. 69, No. 10, October 1992
Auspharm InternationalPty Ltd, POBox 151,Jesmond, New South Wales 2299 Branson Sonic Power Co, Danbury, CT USA ACA Ultragel 22 LKB, Uppsala, Sweden
255
with a history of freedom fromM hyopneumoniae infection. The pigs were transferred to a research piggery at the Veterinary Laboratories, Glenfield where they were fed a commercial grower diet free from antibiotics.
Production of Hyperimmune Sera M hyopneumoniae (J strain), M hyorhinis (GDL strain) and Mflocculare (MS42) were cultured in 800 ml broth (Friis 1977) and washed 3 times in TBS as previously described After the final centrifugation, each pellet was made up to 6 ml with TBS and dispensed in 2 ml volumes. Two ml of Freund's complete adjuvant' was added and the solution emulsified. Pigs were injected intramuscularly with 4 ml of the emulsion and the injection was repeated 2 and 4 wk later. Blood samples were collected 2 wk after the third immunisation. Sera from these pigs were used to establish the appropriate working dilutions for the ELISA.
M hyopneumoniae Challenge Infection Nine pigs were challenged intratracheally with a virulent strain of M hyopnzurmniae (Beaufort strain) originally isolated from a commercial piggery in Victoria (Etheridge et uf 1979) and obtained from A Pointon as lung homogenate in antibiotic-free M hyopneumoniae medium (Friis 1977). The pigs were sedated with azaperone (5 ml)** and placed under general anaesthesia using a face mask with halothanett and nitrous oxide. An 1 .51
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Collection of Tissues and Serafrom Selected Commercial Herds Herds were classified as endemic or free from M hyopneumoniae infection based on prior abattoir monitoring for lung lesions at slaughter, and long-term clinical monitoring of the herds by field veterinarians. A total of 261 blood samples were collected from four herds free from M hyopneumoniae pneumonia. From a further five herds classified as infected with M hyopneumoniae 92 sera were collected from pigs observed to have pneumonic lesions at slaughter. At slaughter, blood samples were collected, lungs observed for the presence of pneumonic lesions (Goodwin and Whittlestone 1973) and lung tissue collected for immunofluorescent histopathology (Sheldrake et a1 1990). Control Sera and ELJSA Ratio Determination Sera were obtained from a number of animals from those herds known to be free from enzootic pneumonia, described previously. The sera were pooled, dispensed in volumes of 50 pl and stored at -70' C. A sample of this pooled control negative serum, was applied to each plate in quadruplicate. After absorbance determination at 405 nm, the mean for the control negative standard was determined. All test sample absorbance values were divided by the mean control negative serum absorbance and the result referred to as the ELSA rafio. In addition, a control positive serum was applied in quadruplicate to each plate in a similar manner. An ELISA ratio of 5 f 10% was required. If the variation of the positive control exceeded this level the assay for the plate was repeated.
Results Serafrom HyperimmunisedPigs: EUSA Absorbances and Ratios In Figure 1, panel A shows the absorbance of sera from pigs hyperimmunised with M hyopneumoniae and M hyorhinis compared with the pooled control negative serum. In panel B the results are expressed as a ratio of the hyperimmunised sera to the control negative serum. These observations were conductedover arange of serum dilutions. M hyorhinis antiserumreactedpoorly, whereas there was a strong reaction with M hyopneumoniae antiserum. The ELISA ratio forM hyopneumoniae increased with increasing serum dilution.
a
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endotracheal tube was inserted into the trachea, and 10 ml of the lung homogenate infused. Pigs were allowed to recover from the anaesthesia and then placed in an isolated pen within the research piggery. Blood samples were collected from pigs upon arrival and again 60 d after challenge (Sheldrake et a1 1990). At slaughter, 60 to 84 d after challenge, tissue was collected for immunofiuorescent histopathology.
4
w
A 2 1
0,
~
5
10
20
50
100
Reciprocal Serum Dilution
Figure 1. (A)The effectof seturn dilutionon absorbance:., negativeconirol serum; A r"vl hyorhinis hyperimmune serum; B. M hyopneumoniae hyperimmuneserum. (B): The effect of serum dilution on the ELISA ratio for: A ,M hyorhinis hyperimmune serum; M hyopneurnoniae hyperimmune serum. Difco Laboratories, Detroit, Mi, USA '* Stresnim),Boehringer Ingleheim, Sydney, New Sourth Wales tt Fluothane@ICI (Aust) Pty Ltd, Sydney, New South Wales
256
Serum from Infected Pig: ELISA Absorbance and Ratio In Figure 2 serum from a pig after challenge infection with M hyopneumoniae, and the control negative serum are plotted over a range of serum dilutions against absorbance (panel A). In panel B the ratio of the post-challenge absorbances to control negative serum absorbances are shown. At a serum dilution between 1/10 and 1/20 the ratio between the M hyopneumoniae infected serum and the control negative serum was greatest. Effect of Pig Age on ELJSA Ratio There is an increase in the ELISA ratio for pigs aged between 6 and 22 wk and free from pneumonia (Figure 3). However this ratio remains well below the ELISA ratio of 3, recommended as a threshold for classifying animals as infected withM hyopneumoniae,and appears to plateau at aratio of about 1.1 to 1.2. Australian Veterinary Journal Vol. 69,No 10, October 1992
Classifcation of Animals by ELISA The results in Table 1 highlight the distributionof ELISA ratio results from animals of various disease status. The results show a clear difference between those free from disease and those idenflied as affected, as judged by the presence of lung lesions at slaughter. Beyond an ELISA ratio of 3 there were no false positive diagnoses, and below anELISA ratio of 2 there was only 1 false negative diagnosis. Using an ELISA ratio of 3 as a threshold above which animals were categorised as ELISA positive, the sensitivity based on lung pathology was shown to be 95.6%. With an ELISA ratio of 2, below which animals were categorised as ELISA negative, specificity based on lung pathology was 98.8%. Only 7 (2%) animals examined fell within the range 2 to 3 and these results were considered inconclusive.
M flocculare (Table 2). Unfortunately no blood samples from field cases of pigs infected with M hyorhinis or Mflocculare and freefromM hyopneumoniae were available. Giventhat there was little reaction using the sera from hyperimmunised animals, cross-reactionfollowing a field infection with eitherM hyorhinis or M flocculare would be unlikely. Armstrong et al (1987) reportedsimilarresults althoughthe serum dilutionthey used was 1/125. Mori et d (1988) have reported considerable antigenic cross-reactivity among porcine mycoplasmas using Westem blotting, and unpublished data from our laboratory concur. However, at the dilutions chosen in this study cross-reactivity in the ELISA is minimised. I
1.21
T
Comparison of Serafrom Hyperimmunised Animals Sera from animals hyperimmunised with e i t h e r M hyopneumoniae,M hyorhinis or Mflocculare were compared over a range of serum dilutions. The results show minor crossreaction for the pig immunised with M hyorhinis and no crossreaction with Mflocculare at a serum dilution of 1/20 (Table 2).
Discussion The results reported show a specific and sensitive ELISA method for detecting antibody toMhyopneumoniaein pig serum. There was little cross-reaction in the ELISA with antibodies to M hyorhinis or M flocculare using sera at a dilution of 1/20 obtained from pigs hyperimmunised with M hyorhinis and 2.01
1.5 a,
0 K
m
s 8 Q
6
A
8
10
5 12
6 14
4 18
5
11
16
1 2 7 20
2
3 22
Age (wks) Figure 3. The effect of pig age on mean ELISA ratio for sera obtained from a herd free from M hyopneumoniae. Numbers above the x axis indicatethe number of sera assessed at each time point. Bars indicate standard deviation.
.I___
1.0
TABLE 1
a
M hyopneumoniaeEUSA ratios relative to enzootic pneumonia status
\ 0.51
Pigs with Pigs with Pigs with pneumonic pneumonic experimental lesions at lesions at Mhyopeumoniae slaughter and slaughter infectiont confirmed Mhyopneumoniae by IMF*
Variable Pigs without pneumonic lesions
01 5
10
20
50
1 00
Reciprocal Serum Dilution B
71
n*
26 1
13
92
9
ELISA ratio 1.18 Mean
5.52
4.89
4.98
1.53
1.73
1.18
SD
*
0.33
lmmunofluorescence By immunofluorescence 4/9 had confirmed M hyopneumoniae lung lesions at slaughter NO. of pigs TABLE 2 EUSA ratios for hyperimmune sera ELISA ratios at serum dilutions
Antiserum 0
5
10
20
50
115
1/10
1120
1150
Mhyopneumoniae 3.0
4.1
4.8
5.9
6.6
6.1
M hyorhinis
1.7
1.9
2.0
2.2
2.0
1.8
M flocculare
1.0
1.1
1.3
1.0
1.1
1.1
100
Reciprocal Serum Dilution Figure 2. (A) The effect of serum dilution on absorbance for serum from a pig challenged with M hyopneumoniae:H, after challenge; 0 ,control negative serum. (B): The effect of serum dilution on the EUSA ratio; A ratio of serum absorbance after challenge to control negative serum.
Australian VeterinaryJournal Vol. 69,No. 10, October 1992
~~
11100 11200
~
257
ELISA ratios were used rather than absorbance to express the results, with the denominator being the control negative serum. This allows comparison among plates; removing plate-to-plate variation. With ratio thresholds of greater than 3 representing a positive diagnosis, less than 2 representing a negative diagnosis and the region 2 to 3 being considered inconclusive, the ELISA correctly identified all animals without lung lesions. Only one animal observed to have a lung lesion at slaughter had an ELISA ratio less than 2, representing a false negative diagnosis rate of 1%. The inconclusive region with ELISA ratios between 2 and 3 probably represents animals in the early stage of infection, and only 2% of animals examined fell within this region. Recently we have shown that with an heterologous strain of M hyopneumoniae, antibody may be observed in blood 7 to 10 d after challenge (Sheldrake et a1 1990). Moreover, this inconclusiveELISA range may become more accurately defined as the number of animals of different age tested increases. It should be emphasised that interpretation of this ratio is arbitrary, in that it is dependent upon the value of the control negative serum. For routine use, large stocks of serum from negative herds need to be pooled and stored at -7OoC, to ensure uniformity in testing between laboratories. In Figure 3 it can be seen that as pigs in herds free from M hyopneumoniae increase in age, the ELISA ratio increases, although only to a value slightly greater than one. However with an increasing data base it may be possible to include the age of the pigs in the ELISA ratio computation. While several authors have published descriptions of ELISA for M hyopneumoniae antibody in porcine serum, we describe the specificity and sensitivity of the test with respect to a large number of samples from commercial herds. Nicolet e l a1 (1980) used Tween 20 soluble protein from a whole cell suspension and after fractionating with gel chromatography obtained a fraction with multiple molecular weights. This fraction yielded good results, although no comparisonswere made with animals known to be generating antibody to M hyorhinis. Freeman et a1 (1984) showed considerable cross-reactivity between M hyopneumoniae and M flocculare antigens and to a lesser degree somereaction withM hyorhinis, however the antigen used in their ELISA was only a crude cell extract. Our results describe a specific and sensitiveELISA for detecting antibody in pig serum to M hyopneumoniaeusing a commercially available antigen (AuspharmS).This procedure should be readily suited to diagnostic laboratories, and will obviate much of the need for culture and imrnunofluorescent histopathology, reducing the cost of M hyopneumoniae pneumonia diagnosis.
SHORT CONTRIBUTIONS
Perennial ryegrass staggers in fallow deer (Dama dama) Regional Veterinary Laboratory, Department of Agriculture, PO Box 483, Baimsdale, Victoria 3875 Department of Agriculture, 51 McCartin St, Lwngatha, Victoria 3953
PJ MRCHELL
CJ McCAUGHAN
Ryegrass staggers is a disease caused by the ingestion of tremorgenic toxins present in perennial ryegrass (Lolium perenne) infested with fungal endophytes (Fletcher and Harvey
258
Acknowledgments We are grateful toDr APointon, Central Veterinary Laboratory, Adelaide, for providing strains of Mhyopnezunoniae,M hyorhinis and Mflocculare, and the rabbit antisera to these organisms. We thank Mrs M M Saunders for her excellent technical assistance and Messrs A Riley and B Philpott who assisted with the pig trials. We are grateful to the Australian Pig Research and Development Corporation,The NSW SwineCompensationFund and Auspharm International Pty Ltd for financial assistance.
References Armstrong C H. Sands-Freeman L and Freeman M J (1987) Can J Vet Res 51:185 Backstrom Land Hoefling D C (1982) Vet CIinNorth Am Lorge Anint Pract 4:259 Bruggmann S, Reller H, Bertschinger H U and Engberg B (1977) Vet Rec 101:109 Bculanger P and L’Ecuyer C (1968) Can J Comp Med32:547 Eberli A (1987) Inaugural Dissertation zur Erlangung der Doctorwiirde, Faculty of Veterinary Medicine, University of Zurich, Switzerland. Etheridge J R, Cottew G S and Lloyd L C (1979)Aurt Vet J55:356 Etheridge J R and Lloyd L C (1980)Aurt Vet J56101 Feld N C, Qvist P, Ahrens P, Friis N F and Meyling A (1990) Proc 8th CongressInternationalOrganisaFat ionfor Mycoplasmology,Istanbul,p 375 Freeman M J. Armstrong C H, Sands-Freeman L L and Lopez-Osuna M (1984) Can J Comp Med 48:202 Friis N F (1977) Acta Vet Scand 18:168 Goodwin R F W. Hodgson R G, Whittlestone P and Woodhams R L (1969) J Hyg 67:193 Goodwin R F Wand Whittlestone P (1973)Br Vet J129456 Kazama S, Yagihashi T and Seto K (1989) Can J Vet Res 53:176 Kobisch M and Nicolet J (1987) Israel J Med Sci 23644 Lam K M and Switzer W P (1971) Am J Vet Res 32: 1737 McKean J D, Andrews J J and Farrington D 0 (1979) J Am Vet Med Assoc 1’74 171 Mori Y.Hamaoka T and Sato S (1987) Israel J Med Sci 23657 Mori Y. Hamaoka T, Sat0 S and Takeuchi S (1988) Vet lmmunol ImmwropaFatholl9239 Muirhead M R (1987) In Respiratory Diseases, University of Sydney Post-graduate Committee in Veterinary Science,ProceedingsNo 95, p571 Nicolet I, Paroz P and Bruggmann S (1980) Res Vet Sci 29:305 Roberts D H and Little T W A (1970)J Comp Path 80:211 Sheldrake R F, Gardner I. Saunders M M and Romalis L F (1990) Aust Vet J 6739
(Acceptedfor publication 29 April 1992)
1981; Gallagher et a1 1982a). The disease occurs in sheep, cattle and horses (Cunningham and Hartley 1959). and has also been reported in wapiti and red deer (different sub-species of Cervus elaphus) in New Zealand (Mackintosh et a1 1982; Brooks and Cahill 1985). Morbidity may be high, but mortality is low (Cunningham and Hartley 1959) and is usually due to misadventure or starvation in severely affected animals. Low weight gains and low concentrations of reproductive hormones are also associated with the disease (Peterson et a1 1984; Fletcher and Barrel1 1984). Fallow deer (Duma dama) were imported to Victoria and Tasmania before 1840 (English 1984a). Only small numbers remain in the wild, mainly in Tasmania, but increasing numbers are being farmed. The number on farms in Australia increased from 26 600 in 1986 to 51 O00 (49.5% of all farmed deer) in 1990 (Coombs 1990); in Victona, they comprise 69.1% of farmed deer (Anon 1991).
Australian Veterinary Journal Vol. 69, No 10, October 1992
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Introduction Enzootic pneumonia caused by Mycoplasma hyopneumoniae is a disease of major economic importance to pig production worldwide (Backstrom andHoefling 1982;Muirhead 1987). One of the difficulties in effectively controlling the disease has been the lack of a simple and cost effective means of monitoring progress of the disease in the herd. Complement fixation tests (Boulanger and L'Ecuyer 1968; Roberts and Little 1970; Etheridge and Lloyd 1980) and indirect haemagglutination tests (Goodwin et a1 1969; Lam and Switzer 1971) have been used although problems with specificity and sensitivity have been reported (McKean et al 1979). More recently enzyme-linked immunosorbent assays (ELISA) have been used (Bruggmann et al 1977; Nicolet et a1 1980; Freemanet a1 19W, Kobisch andNicolet 1987;Eberli 1987; Mori et a1 1987; ). The antigens that were used in these assays were prepared according to the methods of Bruggmann et a1 (1977) or Nicolet et uZ(1980) by the addition of a solubilising detergent to a suspension of whole cells that was semi-purified by column chromatography. Kazama et a1 (1989) used a Tween 20 solubilised fraction, which was prepared by molecular sievingt. These workers found superior specificity compared with the method of Nicolet el a1 (1980). Feld et a1 (1990) evaluated a monoclonal blocking ELISA and found high specificity when compared with an indirect haemagglutination assay. Our aim was to evaluate an ELISA that used solubilised and purified antigens, and to determine the optimum conditions under which the ELISA might be used as a routine diagnostic test.
Materials and Methods Antigens The antigens used in the ELISA were prepared according to the procedures of Auspharm International* and are commercially available through Auspharm. They were obtained from a culture ofM hyopneumoniae (J Strain) which was cultured in broth (Friis 1977) by rotating continuously at 37°C until the change in pH resulted in the medium being honey coloured (pH 6.9 - 7.2). The broth culture was centrifuged at 10 000 g for 20 min. The pellet was subsequently washed 3 times in Tris buffered saline (TBS) (pH 7.4) in a similar manner. After the fmal wash the pellet was resuspended in 10 ml TBS, and sonicated for 30 s in a sonicatorO using a 50% duty cycle, output 3.5. The sonicate was then centrifuged and the pellet Collected and resuspended in TBS. After equilibration in TBS containing 0.2% sodium dodec 1 sulphate (SDS) the material was further purified on a column .
!?
The peak collected between 360 and 405 ml was pooled and concentrated. The antigen was titrated from 1/50 to 1/2OOO and at a titration of 1/1000 the ELISA reaction maximised the response for sera dilutions ranging from 1/20 to 1/250. The antigen was applied to the ELISA plate in a volume of 100 11. The procedures for coating and reading the plates have been described previously (Sheldrake el al 1990).
Animals To provide hyperimmune serum, and serum before and after infection with M hyopneumoniae, six Landrace x Large White pigs6 to 8weeksofagewereobtainedfromacomercialpigge1y
* Present address: Division of Animal Industries, NSW Agriculture, Locked Bag 21, Orange, New South Wales 2800 Sephacryl@,Pharmacia Chemicals, Copenhagen, Denmark
Australian Veterinary JOWMI Vol. 69, No. 10, October 1992
Auspharm InternationalPty Ltd, POBox 151,Jesmond, New South Wales 2299 Branson Sonic Power Co, Danbury, CT USA ACA Ultragel 22 LKB, Uppsala, Sweden
255
with a history of freedom fromM hyopneumoniae infection. The pigs were transferred to a research piggery at the Veterinary Laboratories, Glenfield where they were fed a commercial grower diet free from antibiotics.
Production of Hyperimmune Sera M hyopneumoniae (J strain), M hyorhinis (GDL strain) and Mflocculare (MS42) were cultured in 800 ml broth (Friis 1977) and washed 3 times in TBS as previously described After the final centrifugation, each pellet was made up to 6 ml with TBS and dispensed in 2 ml volumes. Two ml of Freund's complete adjuvant' was added and the solution emulsified. Pigs were injected intramuscularly with 4 ml of the emulsion and the injection was repeated 2 and 4 wk later. Blood samples were collected 2 wk after the third immunisation. Sera from these pigs were used to establish the appropriate working dilutions for the ELISA.
M hyopneumoniae Challenge Infection Nine pigs were challenged intratracheally with a virulent strain of M hyopnzurmniae (Beaufort strain) originally isolated from a commercial piggery in Victoria (Etheridge et uf 1979) and obtained from A Pointon as lung homogenate in antibiotic-free M hyopneumoniae medium (Friis 1977). The pigs were sedated with azaperone (5 ml)** and placed under general anaesthesia using a face mask with halothanett and nitrous oxide. An 1 .51
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0.5
&
0
0
.-~
~~
5
10
20
50
100
Reciprocal Serum Dilution
6
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c
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Collection of Tissues and Serafrom Selected Commercial Herds Herds were classified as endemic or free from M hyopneumoniae infection based on prior abattoir monitoring for lung lesions at slaughter, and long-term clinical monitoring of the herds by field veterinarians. A total of 261 blood samples were collected from four herds free from M hyopneumoniae pneumonia. From a further five herds classified as infected with M hyopneumoniae 92 sera were collected from pigs observed to have pneumonic lesions at slaughter. At slaughter, blood samples were collected, lungs observed for the presence of pneumonic lesions (Goodwin and Whittlestone 1973) and lung tissue collected for immunofluorescent histopathology (Sheldrake et a1 1990). Control Sera and ELJSA Ratio Determination Sera were obtained from a number of animals from those herds known to be free from enzootic pneumonia, described previously. The sera were pooled, dispensed in volumes of 50 pl and stored at -70' C. A sample of this pooled control negative serum, was applied to each plate in quadruplicate. After absorbance determination at 405 nm, the mean for the control negative standard was determined. All test sample absorbance values were divided by the mean control negative serum absorbance and the result referred to as the ELSA rafio. In addition, a control positive serum was applied in quadruplicate to each plate in a similar manner. An ELISA ratio of 5 f 10% was required. If the variation of the positive control exceeded this level the assay for the plate was repeated.
Results Serafrom HyperimmunisedPigs: EUSA Absorbances and Ratios In Figure 1, panel A shows the absorbance of sera from pigs hyperimmunised with M hyopneumoniae and M hyorhinis compared with the pooled control negative serum. In panel B the results are expressed as a ratio of the hyperimmunised sera to the control negative serum. These observations were conductedover arange of serum dilutions. M hyorhinis antiserumreactedpoorly, whereas there was a strong reaction with M hyopneumoniae antiserum. The ELISA ratio forM hyopneumoniae increased with increasing serum dilution.
a
z
endotracheal tube was inserted into the trachea, and 10 ml of the lung homogenate infused. Pigs were allowed to recover from the anaesthesia and then placed in an isolated pen within the research piggery. Blood samples were collected from pigs upon arrival and again 60 d after challenge (Sheldrake et a1 1990). At slaughter, 60 to 84 d after challenge, tissue was collected for immunofiuorescent histopathology.
4
w
A 2 1
0,
~
5
10
20
50
100
Reciprocal Serum Dilution
Figure 1. (A)The effectof seturn dilutionon absorbance:., negativeconirol serum; A r"vl hyorhinis hyperimmune serum; B. M hyopneumoniae hyperimmuneserum. (B): The effect of serum dilution on the ELISA ratio for: A ,M hyorhinis hyperimmune serum; M hyopneurnoniae hyperimmune serum. Difco Laboratories, Detroit, Mi, USA '* Stresnim),Boehringer Ingleheim, Sydney, New Sourth Wales tt Fluothane@ICI (Aust) Pty Ltd, Sydney, New South Wales
256
Serum from Infected Pig: ELISA Absorbance and Ratio In Figure 2 serum from a pig after challenge infection with M hyopneumoniae, and the control negative serum are plotted over a range of serum dilutions against absorbance (panel A). In panel B the ratio of the post-challenge absorbances to control negative serum absorbances are shown. At a serum dilution between 1/10 and 1/20 the ratio between the M hyopneumoniae infected serum and the control negative serum was greatest. Effect of Pig Age on ELJSA Ratio There is an increase in the ELISA ratio for pigs aged between 6 and 22 wk and free from pneumonia (Figure 3). However this ratio remains well below the ELISA ratio of 3, recommended as a threshold for classifying animals as infected withM hyopneumoniae,and appears to plateau at aratio of about 1.1 to 1.2. Australian Veterinary Journal Vol. 69,No 10, October 1992
Classifcation of Animals by ELISA The results in Table 1 highlight the distributionof ELISA ratio results from animals of various disease status. The results show a clear difference between those free from disease and those idenflied as affected, as judged by the presence of lung lesions at slaughter. Beyond an ELISA ratio of 3 there were no false positive diagnoses, and below anELISA ratio of 2 there was only 1 false negative diagnosis. Using an ELISA ratio of 3 as a threshold above which animals were categorised as ELISA positive, the sensitivity based on lung pathology was shown to be 95.6%. With an ELISA ratio of 2, below which animals were categorised as ELISA negative, specificity based on lung pathology was 98.8%. Only 7 (2%) animals examined fell within the range 2 to 3 and these results were considered inconclusive.
M flocculare (Table 2). Unfortunately no blood samples from field cases of pigs infected with M hyorhinis or Mflocculare and freefromM hyopneumoniae were available. Giventhat there was little reaction using the sera from hyperimmunised animals, cross-reactionfollowing a field infection with eitherM hyorhinis or M flocculare would be unlikely. Armstrong et al (1987) reportedsimilarresults althoughthe serum dilutionthey used was 1/125. Mori et d (1988) have reported considerable antigenic cross-reactivity among porcine mycoplasmas using Westem blotting, and unpublished data from our laboratory concur. However, at the dilutions chosen in this study cross-reactivity in the ELISA is minimised. I
1.21
T
Comparison of Serafrom Hyperimmunised Animals Sera from animals hyperimmunised with e i t h e r M hyopneumoniae,M hyorhinis or Mflocculare were compared over a range of serum dilutions. The results show minor crossreaction for the pig immunised with M hyorhinis and no crossreaction with Mflocculare at a serum dilution of 1/20 (Table 2).
Discussion The results reported show a specific and sensitive ELISA method for detecting antibody toMhyopneumoniaein pig serum. There was little cross-reaction in the ELISA with antibodies to M hyorhinis or M flocculare using sera at a dilution of 1/20 obtained from pigs hyperimmunised with M hyorhinis and 2.01
1.5 a,
0 K
m
s 8 Q
6
A
8
10
5 12
6 14
4 18
5
11
16
1 2 7 20
2
3 22
Age (wks) Figure 3. The effect of pig age on mean ELISA ratio for sera obtained from a herd free from M hyopneumoniae. Numbers above the x axis indicatethe number of sera assessed at each time point. Bars indicate standard deviation.
.I___
1.0
TABLE 1
a
M hyopneumoniaeEUSA ratios relative to enzootic pneumonia status
\ 0.51
Pigs with Pigs with Pigs with pneumonic pneumonic experimental lesions at lesions at Mhyopeumoniae slaughter and slaughter infectiont confirmed Mhyopneumoniae by IMF*
Variable Pigs without pneumonic lesions
01 5
10
20
50
1 00
Reciprocal Serum Dilution B
71
n*
26 1
13
92
9
ELISA ratio 1.18 Mean
5.52
4.89
4.98
1.53
1.73
1.18
SD
*
0.33
lmmunofluorescence By immunofluorescence 4/9 had confirmed M hyopneumoniae lung lesions at slaughter NO. of pigs TABLE 2 EUSA ratios for hyperimmune sera ELISA ratios at serum dilutions
Antiserum 0
5
10
20
50
115
1/10
1120
1150
Mhyopneumoniae 3.0
4.1
4.8
5.9
6.6
6.1
M hyorhinis
1.7
1.9
2.0
2.2
2.0
1.8
M flocculare
1.0
1.1
1.3
1.0
1.1
1.1
100
Reciprocal Serum Dilution Figure 2. (A) The effect of serum dilution on absorbance for serum from a pig challenged with M hyopneumoniae:H, after challenge; 0 ,control negative serum. (B): The effect of serum dilution on the EUSA ratio; A ratio of serum absorbance after challenge to control negative serum.
Australian VeterinaryJournal Vol. 69,No. 10, October 1992
~~
11100 11200
~
257
ELISA ratios were used rather than absorbance to express the results, with the denominator being the control negative serum. This allows comparison among plates; removing plate-to-plate variation. With ratio thresholds of greater than 3 representing a positive diagnosis, less than 2 representing a negative diagnosis and the region 2 to 3 being considered inconclusive, the ELISA correctly identified all animals without lung lesions. Only one animal observed to have a lung lesion at slaughter had an ELISA ratio less than 2, representing a false negative diagnosis rate of 1%. The inconclusive region with ELISA ratios between 2 and 3 probably represents animals in the early stage of infection, and only 2% of animals examined fell within this region. Recently we have shown that with an heterologous strain of M hyopneumoniae, antibody may be observed in blood 7 to 10 d after challenge (Sheldrake et a1 1990). Moreover, this inconclusiveELISA range may become more accurately defined as the number of animals of different age tested increases. It should be emphasised that interpretation of this ratio is arbitrary, in that it is dependent upon the value of the control negative serum. For routine use, large stocks of serum from negative herds need to be pooled and stored at -7OoC, to ensure uniformity in testing between laboratories. In Figure 3 it can be seen that as pigs in herds free from M hyopneumoniae increase in age, the ELISA ratio increases, although only to a value slightly greater than one. However with an increasing data base it may be possible to include the age of the pigs in the ELISA ratio computation. While several authors have published descriptions of ELISA for M hyopneumoniae antibody in porcine serum, we describe the specificity and sensitivity of the test with respect to a large number of samples from commercial herds. Nicolet e l a1 (1980) used Tween 20 soluble protein from a whole cell suspension and after fractionating with gel chromatography obtained a fraction with multiple molecular weights. This fraction yielded good results, although no comparisonswere made with animals known to be generating antibody to M hyorhinis. Freeman et a1 (1984) showed considerable cross-reactivity between M hyopneumoniae and M flocculare antigens and to a lesser degree somereaction withM hyorhinis, however the antigen used in their ELISA was only a crude cell extract. Our results describe a specific and sensitiveELISA for detecting antibody in pig serum to M hyopneumoniaeusing a commercially available antigen (AuspharmS).This procedure should be readily suited to diagnostic laboratories, and will obviate much of the need for culture and imrnunofluorescent histopathology, reducing the cost of M hyopneumoniae pneumonia diagnosis.
SHORT CONTRIBUTIONS
Perennial ryegrass staggers in fallow deer (Dama dama) Regional Veterinary Laboratory, Department of Agriculture, PO Box 483, Baimsdale, Victoria 3875 Department of Agriculture, 51 McCartin St, Lwngatha, Victoria 3953
PJ MRCHELL
CJ McCAUGHAN
Ryegrass staggers is a disease caused by the ingestion of tremorgenic toxins present in perennial ryegrass (Lolium perenne) infested with fungal endophytes (Fletcher and Harvey
258
Acknowledgments We are grateful toDr APointon, Central Veterinary Laboratory, Adelaide, for providing strains of Mhyopnezunoniae,M hyorhinis and Mflocculare, and the rabbit antisera to these organisms. We thank Mrs M M Saunders for her excellent technical assistance and Messrs A Riley and B Philpott who assisted with the pig trials. We are grateful to the Australian Pig Research and Development Corporation,The NSW SwineCompensationFund and Auspharm International Pty Ltd for financial assistance.
References Armstrong C H. Sands-Freeman L and Freeman M J (1987) Can J Vet Res 51:185 Backstrom Land Hoefling D C (1982) Vet CIinNorth Am Lorge Anint Pract 4:259 Bruggmann S, Reller H, Bertschinger H U and Engberg B (1977) Vet Rec 101:109 Bculanger P and L’Ecuyer C (1968) Can J Comp Med32:547 Eberli A (1987) Inaugural Dissertation zur Erlangung der Doctorwiirde, Faculty of Veterinary Medicine, University of Zurich, Switzerland. Etheridge J R, Cottew G S and Lloyd L C (1979)Aurt Vet J55:356 Etheridge J R and Lloyd L C (1980)Aurt Vet J56101 Feld N C, Qvist P, Ahrens P, Friis N F and Meyling A (1990) Proc 8th CongressInternationalOrganisaFat ionfor Mycoplasmology,Istanbul,p 375 Freeman M J. Armstrong C H, Sands-Freeman L L and Lopez-Osuna M (1984) Can J Comp Med 48:202 Friis N F (1977) Acta Vet Scand 18:168 Goodwin R F W. Hodgson R G, Whittlestone P and Woodhams R L (1969) J Hyg 67:193 Goodwin R F Wand Whittlestone P (1973)Br Vet J129456 Kazama S, Yagihashi T and Seto K (1989) Can J Vet Res 53:176 Kobisch M and Nicolet J (1987) Israel J Med Sci 23644 Lam K M and Switzer W P (1971) Am J Vet Res 32: 1737 McKean J D, Andrews J J and Farrington D 0 (1979) J Am Vet Med Assoc 1’74 171 Mori Y.Hamaoka T and Sato S (1987) Israel J Med Sci 23657 Mori Y. Hamaoka T, Sat0 S and Takeuchi S (1988) Vet lmmunol ImmwropaFatholl9239 Muirhead M R (1987) In Respiratory Diseases, University of Sydney Post-graduate Committee in Veterinary Science,ProceedingsNo 95, p571 Nicolet I, Paroz P and Bruggmann S (1980) Res Vet Sci 29:305 Roberts D H and Little T W A (1970)J Comp Path 80:211 Sheldrake R F, Gardner I. Saunders M M and Romalis L F (1990) Aust Vet J 6739
(Acceptedfor publication 29 April 1992)
1981; Gallagher et a1 1982a). The disease occurs in sheep, cattle and horses (Cunningham and Hartley 1959). and has also been reported in wapiti and red deer (different sub-species of Cervus elaphus) in New Zealand (Mackintosh et a1 1982; Brooks and Cahill 1985). Morbidity may be high, but mortality is low (Cunningham and Hartley 1959) and is usually due to misadventure or starvation in severely affected animals. Low weight gains and low concentrations of reproductive hormones are also associated with the disease (Peterson et a1 1984; Fletcher and Barrel1 1984). Fallow deer (Duma dama) were imported to Victoria and Tasmania before 1840 (English 1984a). Only small numbers remain in the wild, mainly in Tasmania, but increasing numbers are being farmed. The number on farms in Australia increased from 26 600 in 1986 to 51 O00 (49.5% of all farmed deer) in 1990 (Coombs 1990); in Victona, they comprise 69.1% of farmed deer (Anon 1991).
Australian Veterinary Journal Vol. 69, No 10, October 1992
